The inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate is enhanced by EDTA and diminished by zinc(II)

The sensitivity of the Mg(II)-dependent activity of rabbit liver fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) to inhibition by fructose 2,6-bisphosphate (Fru-2,6-P2) was enhanced by EDTA and diminished to negligible levels by 0.5-2 microM Zn(II) added as another FBPase inhibitor. Fru-2,6-P2 was more efficient in the presence of the synergistic effector AMP: still, the Fru-2,6-P2 concentration inhibiting 50% changed from 3 microM (with EDTA) to higher than 50 microM (with Zn(II]. On the other hand, the Zn(II)-dependent FBPase activity was inhibited by Fru-2,6-P2 to a much lesser extent than the Mg(II)-dependent activity.     

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 μM, 50% inhibition was observed at 4.8 μM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (K_j = 16 μM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate-fructose-1,6-bisphosphatase active site interaction.     

Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Rat liver fructose-1,6-bisphosphatase, which was assayed by measuring the release of 32P from fructose 1,6-[1-32P]bisphosphate at pH 7.5, exhibited hyperbolic kinetics with regard to its substrate. beta-D-Fructose 2,6-bisphosphate, an activator of hepatic phosphofructokinase, was found to be a potent inhibitor of the enzyme. The inhibition was competitive in nature and the Ki was estimated to be 0.5 microM. The Hill coefficient for the reaction was 1.0 in the presence and absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate also enhanced inhibition of the enzyme by the allosteric inhibitor AMP. The possible role of fructose 2,6-bisphosphate in the regulation of substrate cycling at the fructose-1,6-bisphosphatase step is discussed.     

Interaction of fructose 2,6-bisphosphate and AMP with fructose-1,6-bisphosphatase as studied by nuclear magnetic resonance spectroscopy

The interaction of AMP and fructose 2,6-bisphosphate with rabbit liver fructose-1,6-bisphosphatase has been investigated by proton nuclear magnetic resonance spectroscopy (1H NMR). The temperature dependence of the line widths of the proton resonances of AMP as a function of fructose-1,6-bisphosphatase concentration indicates that the nucleotide C2 proton is in fast exchange on the NMR time scale while the C8 proton is exchange limit. The exchange rate constant, koff, has been calculated for the adenine C8 proton and is 1900 s-1. Binding of fructose 6-phosphate and inorganic phosphate, or the regulatory inhibitor, fructose 2,6-bisphosphate, results in a decrease in the dissociation rate constant for AMP from fructose-1,6-bisphosphatase, as indicated by the sharpened AMP signals. A temperature dependence experiment indicates that the AMP protons are in slow exchange when AMP dissociates from the ternary complex. The rate constant for dissociation of AMP from the enzyme.AMP.fructose 2,6-bisphosphate complex is 70 s-1, 27-fold lower than that of AMP from the binary complex. These results are sufficient to explain the enhanced binding of AMP in the presence of fructose 2,6-bisphosphate and, therefore, the synergistic inhibition of fructose-1,6-bisphosphatase observed with these two regulatory ligands. Binding of fructose 2,6-bisphosphate to the enzyme results in broadening of the ligand proton signals. The effect of AMP on the binding of fructose 2,6-bisphosphate to the enzyme has also been investigated. An additional line width broadening of all the fructose 2,6-bisphosphate protons has been observed in the presence of AMP. The assignment of these signals to the sugar was accomplished by two-dimensional proton-proton correlated spectra (two-dimensional COSY) NMR. From these data, it is concluded that AMP can also affect fructose 2,6-bisphosphate binding to fructose-1,6-bisphosphatase.     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

Lysine 274 is essential for fructose 2,6-bisphosphate inhibition of fructose-1,6-bisphosphatase.

Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

The interaction of fructose 2,6-bisphosphate and AMP with rat hepatic fructose 1,6-bisphosphatase

The binding of the inhibitory ligands fructose 2,6-bisphosphate and AMP to rat liver fructose 1,6-bisphosphatase has been investigated. 4 mol of fructose-2,6-P2 and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-bisphosphate exhibits negative cooperatively as indicated by K'1 greater than K'2 greater than K'3 greater than or equal to K'4 and a Hill plot, the curvature of which indicates K'2/K'1 less than 1, K'3/K'2 less than 1, and K'4/K'3 = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K'1 less than K'2 less than K'3 less than K'4 and an nH of 2.05. Fructose 2,6- and fructose 1,6-bisphosphates enhance the binding of AMP as indicated by an increase in the intrinsic association constants. At pH 9.2, where fructose 2,6-bisphosphate and AMP inhibition of the enzyme are diminished, fructose 2,6-bisphosphate binds with a lower affinity but in a positively cooperative manner, whereas AMP exhibits half-sites reactivity with only 2 mol of AMP bound per mol of tetramer. Ultraviolet difference spectroscopy confirmed the results of these binding studies. The site at which fructose 2,6-bisphosphate binds to fructose 1,6-bisphosphatase has been identified as the catalytic site on the basis of the following. 1) Fructose 2,6-bisphosphate binds with a stoichiometry of 1 mol/mol of monomer; 2) covalent modification of the active site with acetylimidazole inhibits fructose 2,6-bisphosphate binding; and 3) alpha-methyl D-fructofuranoside-1,6-P2 and beta-methyl D-fructofuranoside-1,6-P2, substrate analogs, block fructose 2,6-bisphosphate binding. We propose that fructose 2,6-bisphosphate enhances AMP affinity by binding to the active site of the enzyme and bringing about a conformational change which may be similar to that induced by AMP interaction at the allosteric site.     

The regulation of the interaction between F-actin and muscle fructose 1,6-bisphosphatase

Interaction between rabbit muscle fructose 1,6-bisphosphatase (FBPase) and rabbit muscle F-actin results in heterologous complex formation [A. Gizak, D. Rakus, A. Dzugaj, Histol. Histopathol. 18 (2003) 135]. Calculated on the basis of co-sedimentation-binding experiments and ELISA assay-binding constant (Ka) revealed that FBPase binds to F-actin with Ka equal to 7.4 x 10(4) M(-1). The binding is down-regulated by ligands interacting with the FBPase active site (fructose 6-phosphate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate) and with the FBPase allosteric inhibitory site (AMP). The binding and the kinetic data suggests that FBPase may bind F-actin using a bipartite motif which includes the amino acids residues involved in the binding of the substrate as well as of the allosteric inhibitor of the enzyme. The in situ co-localization experiment, in which FBPase was diffused into skinned muscle fibres pre-incubated with phalloidin (polymeric actin-interacting toxin), has shown that FBPase binds predominantly to the region of the Z-line.     

The reactive cysteine residue of pig kidney fructose 1,6-bisphosphatase is related to a fructose 2,6-bisphosphate allosteric site

Modification of a highly reactive cysteine residue of pig kidney fructose 1,6-bisphosphatase with N-ethylmaleimide results in the loss of activation of the enzyme by monovalent cations. Low concentrations of fructose 2,6-bisphosphate or high (inhibitory) levels of fructose 1,6-bisphosphate protect the enzyme against the loss of monovalent cation activation, while non-inhibitory concentrations of the substrate gave partial protection. The allosteric inhibitor AMP markedly increases the reactivity of the cysteine residue. The results indicate that fructose 2,6-bisphosphate can protect the enzyme against the loss of potassium activation by binding to an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit the enzyme by binding to this allosteric site.     

Antagonistic effects of hypertrehalosemic neuropeptide on the activities of 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase in cockroach fat body

Hypertrehalosemic neuropeptides from the corpora cardiaca such as the decapeptide Bld HrTH bring about a profound switch in the metabolic activity of cockroach fat body during which production of the blood sugar trehalose is stimulated while the catabolism of carbohydrate (glycolysis) is inhibited. The mechanisms of the metabolic switch are not fully understood.Incubation of isolated fat body from the cockroach Blaptica dubia with 10(-8) M Bld HrTH, for 10-60 min, stimulated glycogen breakdown and increased the content of the substrates of both the glycolytic enzyme 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) in the tissue. The glycolytic signal fructose 2,6-bisphosphate was markedly decreased in fat body on incubation with Bld HrTH. The content of ATP was slightly reduced, while the contents of ADP and AMP were increased after incubation with the hormone.Fructose 2,6-bisphosphate is a potent activator of PFK and a strong inhibitor of FBPase purified from fat body. The activity of PFK was decreased by about 90% when the hormone-dependent changes in effectors and substrates in fat body were simulated in vitro. FBPase, in contrast, was activated about 25-fold under these conditions, suggesting the hormone to stimulate gluconeogenesis in fat body. The data support the view that fructose 2,6-bisphosphate is a pivotal intracellular messenger in the hormone-induced metabolic switch from carbohydrate degradation to trehalose production in cockroach fat body.     

Effects of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate on phosphofructokinase from chicken erythrocytes

1. Phosphofructokinase (EC 2.7.1.11) from chicken erythrocytes is activated by fructose 2,6-bisphosphate, glucose 1,6-bisphosphate and AMP, and it is inhibited by 2,3-bisphosphoglycerate and inositol hexaphosphate. 2. The stimulatory effects produced by the two bisphosphorylated hexoses are additive and the effects produced by fructose 2,6-bisphosphate and by AMP are synergistic. 3. The activatory effect produced by fructose 2,6-bisphosphate is counteracted by fructose 1,6-bisphosphate. 4. The inhibition produced by both 2,3-bisphosphoglycerate and inositol hexaphosphate is released by fructose 2,6-bisphosphate. 5. It is concluded that, like phosphofructokinase from mammalian tissues, the enzyme from chicken erythrocytes can be modulated by the relative concentrations of those metabolites.     

Effects of fructose 1,6-bisphosphate on the activation of yeast phosphofructokinase by fructose 2,6-bisphosphate and AMP

Fructose 1,6-bisphosphate decreases the activation of yeast 6-phosphofructokinase (ATP:fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) by fructose 2,6-bisphosphate, especially at cellular substrate concentrations. AMP activation of the enzyme is not influenced by fructose 1,6-bisphosphate. Inorganic phosphate increases the activation by fructose 2,6-bisphosphate and augments the deactivation of the fructose 2,6-bisphosphate activated enzyme by fructose 1,6-bisphosphate. Because various states of yeast glucose metabolism differ in the levels of the two fructose bisphosphates, the observed interactions might be of regulatory significance.     

Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases

Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.     

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.     

The effect of fructose 2,6-bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase from rat liver

Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,6-bisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-1,6-bisphosphatase was more easily inhibited by both effectors. Fructose 2,6-bisphosphate affected both K0.5 and Vmax, while the main effect of AMP was to lower Vmax. Fructose 2,6-bisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation.     

Biochemical characterization of cytosolic fructose-1,6-bisphosphatase from apple (Malus domestica) leaves

Cytosolic fructose-1,6-bisphosphatase was purified to apparent homogeneity from the leaves of apple, a sorbitol synthesizing species. The enzyme was a homotetramer with a subunit mass of 37 kDa, and was highly specific for fructose 1,6-bisphosphate (F1,6BP) with a Km of 3.1 micro M and a Vmax of 48 units (mg protein)(-1). Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mM and 62 micro M, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+ activated enzyme activity. Fructose 6-phosphate (F6P) was found to be a mixed type inhibitor with a Ki of 0.47 mM. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. AMP was a non-competitive inhibitor for the enzyme. F6P interacted with F2,6BP and AMP in a synergistic way to inhibit the enzyme activity. Dihydroxyacetone phosphate slightly inhibited the enzyme activity in the presence or absence of F2,6BP. Sorbitol increased the susceptibility of the enzyme to the inhibition by high concentrations of F1,6BP. High concentrations of sorbitol in the reaction mixture led to a reduction in the enzyme activity.     

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.